ESR data obtained from spin-labeled animal cell membranes support the hypothesis that the inner and outer faces of the cytoplasmic membrane and the intracysternal and extracysternal monolayers of the endoplasmic reticulum bilayer have different phase transition properties. Physical evidence for structural asymmetry in cytoplasmic membranes was corroborated by companion data from physiological studies of several membrane associated functions. Using the physical data, phase boundaries were tentatively established for two lipid compartments of equal size; using the phyiological data, phase boundary temperatures were predicted for the inner and outer monolayers of the cytoplasmic membrane. Future experiments will be designed to elucidate the exact nature of membrane lipid asymmetry with emphasis on the respective roles of phospholipid side chains (i.e., saturated vs. unsaturated fatty acid content), polar head groups,and cholesterol. To elicit the kinds of information needed to establish the physical properties of each monolayer, membrane-enveloped viruses and phagocytized latex beads (phagosomes) derived from a common cell line will be used as sources of right side out and inside out membranes for spin-labeling studies. Methods are also available for obtaining "right side out" endoplasmic reticulum membranes. Treating the membrane samples with nickel and cobalt ions (divalent) should rapidly destroy ESR signals from the outer monolayer, leaving the characteristic temperatures of the inner monolayer intact. Likewise, choline phosphatides of defined fatty acid composition, and agents such as filipin, amphotericin and digitonin (which stoichiometrically bind to cholesterol) will be exchanged into or onto the surface monolayer of membranes to specifically alter the characteristic temperatures of the single exposed monolayer. Membranes from different organelles, tissues, and species will be examined to obtain an overall view of membrane lipid physical asymmetry.